Combinations of scleroderma hallmark autoantibodies associate with distinct clinical phenotypes

Systemic sclerosis (SSc) is characterized by the presence of SSc-specific or SSc-associated antibodies (SSc-Abs): anti-topoisomerase I (ATA), anti-centromere (ACA), anti-RNA polymerase III (ARA), anti-U3RNP (U3RNP), anti-U1RNP (U1RNP), anti-PmScl (PmScl), anti-Ku (Ku) and anti-Th/To (Th/To), each being associated with specific clinical features and prognosis. The detection of more than one SSc-Abs in SSc patients is rare and only few data about these patients’ clinical phenotype is available. The aim of our study was to evaluate the frequency and the disease’s features associated with the presence of > 1 SSc-Abs positivity in a large cohort of SSc patients. The autoantibody profiles of 2799 SSc patients from February 2001 to June 2017 were retrospectively reviewed. Patients with > 1 SSc-Abs were identified. Clinical features were collected and compared to a large historical cohort of SSc patients with single SSc-Ab positivity. SSc patients were excluded if previously treated with rituximab, intravenous immunoglobulins or stem cell transplantation. Non-parametric tests were used for statistical analysis. Nearly 5% of SSc patients from our cohort had ≥ 2 autoantibody positivity, and 2.3% (n = 72) had ≥ 2 SSc-Abs positivity. Th e most common combination was U1RNP and ATA (35%). These patients were younger than patients with single autoantibody positivity and showed more commonly a diffuse cutaneous SSc form. They also had higher rates of overlap features compared to ATA patients. Other combinations included U1RNP and ACA (13%), ATA and ACA (7%) and U1RNP and PmScl (5%). In our study we observed that, while infrequently, SSc patients can present with a combination of two SSc-Abs and that the double positivity can influence their clinical phenotype compared to patients with single SSc-Ab positivity. The importance of re-testing SSc-Abs in patients with changing clinical phenotypes was also highlighted, as this may confer a differing risk stratification.

www.nature.com/scientificreports/ relatively frequent co-existence of non-SSc specific autoantibodies, most commonly anti-Ro (in up to 56% of patients with ACA) 9 .
Traditionally the SSc specific autoantibodies were thought to be mutually exclusive 6,[9][10][11][12] , but there is a small body of literature on their coexistence. The most commonly reported SSc specific dual positivity is the co-existence of ATA and ACA (0.05-0.6% of SSc patients 13,14 ). The clinical phenotype of this combination of autoantibodies seems to mirror the ATA population with 60% of patients being dcSSc, and having clinical and visceral symptoms in keeping with ATA single positivity.
Previous works have not focused on clinical phenotypes of other double SSc specific autoantibody positivity. Therefore, the aim of this study was to determine the frequency and clinical phenotypes of patients with the co-existence of more than one SSc specific autoantibodies from our tertiary referral centre for connective tissue diseases.

Methods
The autoantibody (Ab) profiles of 2799 SSc patients from February 2001 to June 2017 were retrospectively reviewed. All patients met the ACR/EULAR 2013 classification criteria for SSc. Patients with > 1 positivity at any time for the following SSc-specific or SSc-associated antibodies were selected: ACA, ATA, U3RNP, Ku, PmScl, U1RNP, Th/To, ARA. Clinical features were collected for these patients, in particular: age, sex, presence of overlap features, SSc subtype, inflammatory arthritis, lung fibrosis on computer tomography (CT), pulmonary arterial hypertension (PAH), scleroderma renal crisis (SRC), cardiac involvement and skeletal myositis. SSc subtype was classified as either lcSSc or dcSSc. Lung fibrosis was defined as the presence of interstitial lung disease (ILD) at CT scan. PAH was diagnosed after right heart catheter in all patients. Cardiac involvement was defined as the presence of diagnostic cardiac biopsy, cardiac magnetic resonance features or the necessity of introduction anti-arrhythmic therapy not explained by other medical conditions. For comparison we included an historical SSc cohort of patients with single positivity for each of the autoantibodies initially selected and followed-up at the same referral centre.
Antibody testing was performed in the hospital immunology laboratory. Antinuclear antibody (ANA) positivity was confirmed by indirect immunofluorescence (IIF) on Hep-2 cells. Positivity for ACA, anti-U3-RNP, and anti-Th/T0 was based on their specific staining pattern. The staining pattern used to define anti-U3-RNP was described by Tormey et al. 15 . The pattern consisted of three elements -clumpy nucleolar staining with staining of the coilin bodies in interphase cells and classic fibrillar or lacy staining in metaphase cells. The staining pattern used to describe anti-Th/T0 was homogeneous staining of the nucleoli with fine speckled staining of the nucleoli with fine speckled staining of the nucleoplasm 16 . The immunology laboratory performed an internal validation of the method, by correlating the pattern observed on indirect immunofluorescence (IIF) and immunoprecipitation (IP) testing, although the results have not been published. Positivity for ARA was confirmed on ELISA. ATA, anti-Pm/Scl, anti-U1-RNP, anti-Ku antibody positivity was tested using counterimmunoelectrophoresis.
Among > 1 antibody positive patients we identified a separate cohort who had a sequential accumulation of antibodies (referred to as "gain antibody cohort"), rather than 2 antibodies from initial testing. For these patients the time interval between additional antibody acquirement was recorded, as well as any change in clinical phenotype. Among the gain antibody patients we then additionally identified patients who had concomitantly lost and acquired a new antibody ("switch antibody cohort").
To avoid drug-related autoantibody misinterpretations, we excluded patients who had been treated with rituximab, intravenous immunoglobulins (IvIg) or stem cell transplantation prior to the immunology tests.
Statistical analysis were performed using SPSS versions 22. Continuous variables are expressed as mean ± standard deviations (SD). Categorical variables are expressed as number (%). Two-sided Fisher's exact test and Mann-Whitney U test were used to perform comparisons. A p value < 0.05 was considered statistically significant.
This study was conducted in compliance with the Declaration of Helsinki. Informed consent was obtained from patients routinely reviewed in our centre which has been approved by the London-Hampstead and the London-Fulham Research Ethics Committees.
Ethics approval and consent to participate. All patients gave consent for their anonymous data to be used.

Results
Study cohort. We initially identified 122 patients (4.3% of our cohort) with greater than one autoantibody where at least one autoantibody was SSc-specific or SSc-associated. We then focused on patients with SSc specific autoantibodies, and identified 72 patients (2.6% of our cohort) with at least two SSc specific autoAb. Full clinical data were available for 63 patients. In particular we found 60 patients (2.1%) with double Ab positivity and 3 patients with triple Ab positivity (0.1%). The antibody most frequently associated with a double positivity was U1RNP (43 patients, 72%), followed by ATA (21 patients 35%) and ACA (19 patients, 32%) (See Table 1). Anti Th/T0 was not found in combination with any of the other antibodies. Interestingly all patients with three autoantibodies present were positive for anti-U1RNP antibody. In our cohort the most frequent combination was U1RNP and ATA (21 patients, 35%) followed by U1RNP and ACA (8 patients, 13%) and U1RNP and U3RNP (6 patients, 10%).
U1RNP and ARA . This group included 4 patients (7%). Compared to U1RNP patients, this cohort had no features of overlap disease (0 vs 53%, p = 0.050) and were more frequently of diffuse cutaneous subtype (75% vs 21%, p = 0.040). Frequency of clinical features were similar compared to patients with each individual autoantibody.
U1RNP and PmScl. This group included 3 patients. The only significant clinical feature was a higher prevalence of skeletal myositis in the double positive patients compared to U1RNP patients (100% vs 16%, p = 0.006).
ATA and ACA . This interesting group included 4 patients (7%). When comparing ATA and ACA patients to ACA patients and ATA patients we found these patients behaved more similarly to ATA than ACA as they had a significantly higher prevalence of lung fibrosis compared to ACA (75% vs 11%, p = 0.006) and of skeletal myositis (25% vs 1%, p = 0.041).
ACA and PmScl. This group included 4 patients. The only significant clinical feature was a higher prevalence of myositis compared to ACA patients (25% vs 1%, p = 0.04).
ACA and U3RNP. This group included only two patients. The only significant clinical feature was a younger age of disease onset compared to ACA patients (51.50 ± 2.12 vs 68.75 ± 12.61, p = 0.045).
Other combinations. No significant clinical features were associated with the other Ab combinations. www.nature.com/scientificreports/ Gain antibody. 32 patients out of 2799 (1.14%) were identified as gaining at least 1 autoantibody during the study period. Of these, 15 patients initially had one SSc specific autoantibody, and gained a second SSc specific autoantibody. One patient gained 2 SSc specific autoantibodies, and 1 patient started with 2 SSc specific Abs and gained a third. The rest gained a non-SSc specific autoantibody and were not included in the subsequent analysis ( Table 4). The most common initial autoantibody was ATA (7 patients 38.9%), followed by U1RNP (4 patients 12.5%). The median time interval between first autoantibody identification and second autoantibody identification was 38 months (range 5-183 months). The most common second Abs were ACA and U1RNP (5 respectively, 29.4%).
The gain of a new SSc specific autoantibody was associated with a clinical modification in 7 patients (38.9%) ( Table 4). 50% of the patients who started with ATA and gained U1RNP developed ILD (interstitial lung disease). In 2 patients the gain in autoantibody was associated with a switch from lcSSc to dcSSc. Switch antibody. A total of 4 patients out of 72 double antibody patients (5%) simultaneously gained and lost an antibody. We have called this group of patients "switch autoantibody" (see Table 5).
Median time interval was 90.5 (77.25 -98.5) months between change in autoantibody. In 3 cases (75%) there was a significant clinical modification of disease' features upon Ab profile modification, in particular one patient developed ILD, one patient had a notable ILD progression, and one patient developed myositis. None of these patients had any medication which could have altered their autoantibody profile.

Discussion
We present our tertiary centre experience of the clinical phenotype of double SSc specific antibody positivity, either at initial testing, or sequential addition of autoantibodies with subsequent testing. We focused only on SSc specific autoantibodies, as they traditionally have been described as being mutually exclusive. There has www.nature.com/scientificreports/ been previous literature discussing double autoantibody with anti-Ro, or anti-La, but these can be non-specific findings, and are more frequently found in combination than the SSc-specific or SSc-associated autoantibodies. Indeed, in a previous study it was found that anti-Ro and/or anti-La positivity were only marginally associated with sicca syndrome and that the positivity for anti-Ro60 was found in strong association with pulmonary fibrosis, but this finding was deemed secondary to the high co-occurrence of anti-Ro60 with ATA 17 . The prevalence of double autoantibody in the literature in all SSc patients ranges from 0.6-5%, with one study reporting up to 35% 18,19 . The larger proportion of SSc double antibody positivity was though not limited to SSc specific autoantibodies, and therefore is likely to account the disproportionate high frequency. Our cohort was comparable with a prevalence of 4.3% having double autoantibody positivity, and 2.3% of our cohort having at least two SSc specific autoantibodies.
Interestingly, we observed not only that the presence of two SSc-specific or SSc-associated autoAbs can influence patients' clinical phenotype but that each autoAb could have a different clinical expression according to the combination observed.
In terms of predominating autoantibody, we found that in the U1RNP and ATA combination, clinical features were more consistent with U1RNP. This was mirrored in the U1RNP and ACA combination, although they had a higher risk of PAH compared to U1RNP on its own suggesting a synergistic effect on the two Abs on the development of PAH. In the U1RNP and PmScl combination, clinical features consistent with PmScl dominated, as did the clinical features of PmScl in the ACA and PmScl combination. In keeping with previous literature, the clinical features of ATA dominated in the double positivity patients of ATA and ACA 14 .
The combination of ACA and ATA has been previously described by numerous cohorts, and ranges between 0.5-5% 13,14,18,19 . Unlike previous studies, the majority of our patients had lcSSc, however they had comparable clinical phenotypes to the single positive ATA patients. Traditionally patients with ATA have a higher frequency Table 3. Comparison between clinical features of double positivity SSc specific autoantibody group, and cohort of patients with single autoantibody positivity. Significant p values (< 0.05) are highlighted in bold. ILD (interstitial lung disease), PAH (pulmonary arterial hypertension), SRC (scleroderma renal crisis).

Double antibody group
Comparator group www.nature.com/scientificreports/ of dcSSc. Other studies have reported increased rates of calcinosis and vascular complications compared to ACA or ATA alone 20 , however our clinical records did not allow for interpretation of this. The most frequent occurring double autoantibody group was U1-RNP and ATA, and within our whole population, made up 0.8% of the patients included in the analysis. These patients were typically younger with higher rate of dcSSc disease. Their clinical phenotype was more comparable to U1RNP than ATA. Consistent with a previous large study not limited to SSc patients, compared to ATA, this combination was more likely to be observed in patients with overlap disease, myositis and arthritis 21 .
ARA and U1 RNP has also previously been reported. In one small case series, 2 patients had SLE, and only 1 had SSc overlap. Unlike this study, none of our patients had overlap features, and the majority of patients had dcSSc 22 , however we only looked at a cohort of patients with established SSc.
Previous studies have focused more on the differing techniques used to identify > 1 autoantibody in the serum of individual patients (immunoblot or ELISA), rather than the clinical phenotypes of the patients with these autoantibody profiles 9,18,23 . All our autoantibody testing was derived from the same laboratory, and utilised the same techniques throughout the study period. This therefore would not explain the sequential gain in autoantibodies, as techniques were not changed in between sample analysis. Discussion on gain SSc specific. It is appreciated that in SSc, autoantibodies remain present throughout the course of the disease, with little variability in titres, except possibly in very mild cases [24][25][26] . The prospective gain of additional SSc specific autoantibodies has very rarely been reported in the literature. In our cohort we identified 15 patients who gained an additional SSc specific autoantibody during the course of their disease, and in 7 of these patients, it was associated with a clinical meaningful change in their disease.
Although it is not our routine practice, these results highlight the potential value of repeat autoantibody testing, especially in the presence of a change in clinical manifestations. Given that this is not routinely done, we do not know the time delay between the gain in the additional SSc specific autoantibody. The number of gain autoantibodies may also be vastly underestimated as many patients may not have had repeat autoantibody testing within the study period. The knowledge of any additional autoantibody is clinically relevant in prognosticating  Table 5. Cohort of patients who initially presented with one autoantibody, and during their condition lost their initial SSc specific Ab, and gained a different one ("switch patients"). The clinical change is also documented. www.nature.com/scientificreports/ patients for potential organ involvement. Further work is needed to decide whether regular testing of autoantibodies would be of merit.
Discussion on switch autoantibodies. There is very little in the literature of the clinical impact of seroconversion of autoantibodies in SSc. In our cohort, 4 patients out of the 72 identified as having more than one SSc specific autoantibody had initially presented with one autoantibody, and switched to another during the course of this disease. In 3 of these patients, a modification of their clinical phenotype was identified. There has been only one case report of a patient converting from anti Th/To to ACA 27 , although neither of these autoantibodies were present in our seroconversion cohort. The seroconversion also predated any change in clinical phenotype. We do not know if this would be the case with our cohort, as autoantibodies are not routinely tested, and a change in the clinical picture may prompt a repeat testing of the autoantibodies.
Limitations. Given the rarity of having two SSc specific autoantibodies, some of the cohorts used in our analysis are very small, and therefore conclusions may not be fully accurate. Moreover, as re-testing of autoantibodies was not routinely performed in our cohort, the real prevalence of double SSc-specific or SSc-associated Abs might be underestimated. This is a retrospective analysis based on immunology results, and available clinical information, which may impact the reliability of all clinically available information.

Conclusion
We present our cohort of patients with at least two SSc specific autoantibodies. Our work highlights that the traditional teaching that these show mutual exclusivity is not wholly accurate, and a small but significant proportion of patients do have more than one SSc specific autoantibody. This double antibody positivity in the majority of patients does confer differing clinical phenotype, either in terms of demographics, or clinical manifestations, compared to those patients with only one of the autoantibodies present. This work also highlights the importance of re-testing immunological profiles with change in clinical phenotypes, as patients may have gained a new autoantibody, and thus their risk stratification profile may also have changed. Prospective studies are needed to identify the patients who will benefit from repeated.

Data availability
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.